The genome of vesicular stomatitis virus is single-stranded RNA which is complementary to its messenger RNA. Transcription of the genome is mediated by an encapsidated transcriptase. The products of transcription are five monocistronic messenger RNAs which are capped and methylated at the 5' end and polyadenylated at the 3' end. Three of the viral encoded polypeptides, L, NS and N, are required and sufficient for these activities. However, their precise roles are not known. In addition, the mechanism by which the genome RNA is replicated is not understood. We are seeking the identity and function of the viral proteins involved in these two pathways of RNA synthesis through analyses of temperature-sensitive mutants which display defective RNA synthesis. The approach is bidirectional: 1) identification of the altered protein subunit in each mutant and 2) determination of the functional consequences of the alterations. In addition, the temperature-sensitive mutants are being used in conjunction with defective interfering particles in attempts to isolate the replicative pathway in vivo and in vitro.